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A DNA-Directed Light-Harvesting/Reaction Center System Palash K Dutta, Symon Levenberg, Andrey Loskutov, Daniel Jun, Rafael Saer, J. Thomas Beatty, Su Lin, Yan Liu, Neal W. Woodbury, and Hao Yan J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja509018g • Publication Date (Web): 23 Oct 2014 Downloaded from http://pubs.acs.org on October 26, 2014

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Journal of the American Chemical Society

A DNA-Directed Light-Harvesting/Reaction Center System Palash K. Dutta,1,2 Symon Levenberg,1,2 Andrey Loskutov,2 Daniel Jun,3 Rafael Saer,3 J. Thomas Beatty,3 Su Lin,1,2 Yan Liu,1,2 Neal W. Woodbury,*,1,2 Hao Yan*,1,2 1

Department of Chemistry and Biochemistry and 2The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, United States 3

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada ABSTRACT: A structurally and compositionally well-defined and spectrally tunable artificial light-harvesting system has been constructed in which multiple organic dyes attached to a 3arm DNA nanostructure serve as an antenna conjugated to a photosynthetic reaction center isolated from Rhodobacter sphaeroides 2.4.1. The light energy absorbed by the dye molecules is transferred to the reaction center where charge separation takes place. The average number of DNA 3arm junctions per reaction center was tuned from 0.75 to 2.35. This DNA-templated multi-chromophore system serves as a modular light-harvesting antenna that is capable of being optimized for its spectral properties, energy transfer efficiency and photo-stability, allowing one to adjust both the size and spectrum of the resulting structures. This may serve as a useful testbed for developing nanostructured photonic systems.

INTRODUCTION: During photosynthesis, light energy is collected by a large light-harvesting network and efficiently transferred to a reaction center (RC), which converts it to chemical energy via charge separation.1 The quantum efficiency of the charge separation reaction by the photosynthetic reaction center is nearly unity.1d The architecture and spectral properties of the light-harvesting system that surrounds the reaction center have evolved to meet the constraints of a broad range

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of different light conditions and environments. A number of researchers have attempted to mimic the natural photosynthetic apparatus by designing artificial light harvesting antenna systems2-5 for a variety of photonic applications.6 To facilitate nanoscale photonic applications more broadly, the construction of artificial antenna systems that provide controllable light absorption, efficient energy transfer and improved photo-stability are desirable. Self-assembling proteins3 and dendrimers4 have been explored to create artificial antenna systems, but they lack a well-defined multi-chromophore geometry and stoichiometry. Synthetic porphyrin structures5 have been investigated to create artificial antennas connected to electron transfer complexes, but these generally have an absorption cross-section that is spectrally relatively narrow. DNA nanotechnology can be used to generate programmable, self-assembled nanostructures7 with multiple fluorophores at welldefined positions, and this approach has been used to create artificial light harvesting antenna systems. Double helical DNA structures, three-way junctions, seven helix bundles and several other DNA based antenna systems8 have been used to create artificial antennas with unidirectional energy transfer along an excited state energy gradient between chromophores that mimics the stepwise energy transfer in some of the natural photosynthetic systems. However, thus far these assemblies have lacked the ability to convert the light energy to redox energy via charge separation. Recently, we have studied different dye molecules directly conjugated to reaction centers and explored the effects of altering the dye spectral and excited state properties on the efficiency of energy transfer and charge-separation9. In this report we go a step further and use a 3armDNA nanostructure to organize multiple dye molecules and specifically assemble these


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nanostructured complexes with reaction centers (Figure 1A), resulting in a geometrically programmable model system mimicking a natural photosynthetic apparatus.

Figure 1. (A) Modified structure of the reaction center (RC) from the purple bacterium, Rhodobacter sphaeroides 2.4.1 (PDB 2J8C

10

) with sequences of the 3arm-DNA construct

shown. The cofactors of the RC are colored and those active in electron transfer reactions involved in this report are designated by letters: P – bacteriochlorophyll pair, BA – bacteriochlorophyll monomer, HA – bacteriopheophytin, QA – ubiquinone. The arrows in the DNA structure point in direction of the 3’ end of the DNA strands. The 3’-Amine modified Strand-1 (purple) of the 3arm-DNA is conjugated to one of the Cys residues (shown in red) on the surface of the RC via a SPDP (N-succinimidyl 3-(2-pyridyldithio) propionate) linker. The other two strands (Strand-2 and -3 in green and red, respectively) are allowed to hybridize to Strand-1 to form the 3arm-DNA junction. Inter-Cys distances on the RC are marked as dotted lines. The two stars on 3arm represent the positions of the two dye molecules, where the cyan


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star corresponds to either Cy3 or AF660, and the pink star corresponds to either Cy5 or AF750. It should be noted that because of the presence of three Cys residues on the surface of the protein, 1 or 2 or 3 copies of Strand-1 can be conjugated to the RC, and consequently up to three 3armDNA junctions (and three pairs of dyes) can be conjugated to the RC. For clarity, only one is shown here. (B) A representative absorption spectrum of RCs that have an average of 2.3 of the 3arm-DNA-Cy3-Cy5 nanostructures attached. (C) An absorbance spectrum of RCs that have an average of 2.1 of the 3arm-DNA-AF660-AF750 nanostructures attached. The absorbance spectra of panels B and C show enhanced absorbance cross-section in the spectral regions 450-700 nm or 500-800 nm, respectively, where the RC absorbance is relatively low. The spectrum of free RC is shown in both panels B and C (red trace) for comparison. Two different pairs of DNA-conjugated chromophores are used in this study: Cy3 and Cy5, or Alexa Fluor 660 and Alexa Fluor 750. Cy3 acts as the donor and Cy5 as the acceptor in the first pair, and AF660 acts as the donor and AF750 as the acceptor in the second pair. The fluorophores were chosen so that there is significant spectral overlap between emission of the dyes and the absorption of the RC to facilitate efficient energy transfer, and so that there is a substantial increase in the absorption cross-section in the spectral regions where the absorbance of the RC alone is low (Figures 1B-C and 3). A very simple 3arm-DNA structure was designed to assemble the two dye molecules in a geometrically defined manner and to avoid chemical modification of any DNA strands with more than one dye (to reduce cost and synthetic complexity) (Figure 1A). Two of the strands (Strand-2 and -3) in the 3arm-DNA contain the dye molecules, and the other one (Strand-1) is conjugated to the RC through a covalent cross-link. The three dimensional structure of the RC complex from Rhodobacter sphaeroides 2.4.1 10

is depicted in Figure 1A, and it consists of three subunits H, M and L. There is a total of ten


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cofactors associated with the L/M transmembrane region of the structure, including a dimer of bacteriochlorophylls

(P),

two

monomer

bacteriochlorophylls

(BA

and

BB),

two

bacteriopheophytins (HA and HB), two ubiquinone-10 molecules (QA and QB), one carotenoid and one nonheme iron (Fe2+).11 The special pair P is the primary donor of electrons in the lightdriven electron transfer process, which subsequently transfers electron to QA via BA and HA, forming a long-lived charge-separated state P+QA-. When ubiquinone is bound in the QB site, electron transfer occurs from QA- to QB forming P+QB-.12 A genetically modified RC was used in these studies and contained a total of eight mutations, five of them to replace the five wild-type cysteines with serine or alanine, and the remaining three to replace three selected wild-type amino acids (asparagine or glutamic acid) with cysteine residues at specific locations on the surface of the RC that are close to the primary electron donor, P.9b,13 Two of the new Cys residues are located on the surface of the L subunit (L72, L274) and the other one is on the surface of the M subunit (M100) (Figure 1A).

RESULTS AND DISCUSSION: Assembly of Light-Harvesting/Reaction Center Complex A 3’-Amine modified Strand-1 was conjugated to the introduced Cys residues of the RC in a 10:1 molar ratio by using a SPDP (N-succinimidyl 3-(2-pyridyldithio) propionate) crosslinker (see details in the Supporting Information). The reaction mixture was subsequently purified by fast protein liquid chromatography (FPLC) (Figure 2) (see Supporting Information for methods). The chromatograph shows four prominent peaks using absorbance at 260 nm and 280 nm, and three peaks using absorbance at 365 nm (Soret peak of RC). The fractions under each peak were collected and characterized. The UV-vis absorbance maxima for the first,


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second and third peaks in the chromatograph are at 271 nm, 268 nm and 266 nm, respectively. The blue shift of the absorbance peak together with a relative increase in the absorbance intensity (compared to the absorbance peak at 800 nm) indicate that the species contained in the peaks have different ratios of DNA conjugated to the RC, increasing from peak 1 to peak 3. (DNA:RC = 1:1, 2:1 and 3:1). It is important to note that the single copy of Strand-1 conjugated to RC can be on any of the three Cys. Similarly, there are three ways that two copies of Strand-1 could be conjugated to the RC. This heterogeneity of the sample is reflected by the widths of the first and second chromatograph peaks. The third peak, in contrast, has the narrowest peak and highest ratio of A260/A365 among the first three and it represents a single species of RC with three copies of Strand-1 conjugated to all of the Cys residues. The last peak in the chromatograph has no absorbance at 365 nm (the Soret absorbance band of the RC), indicating that it is excess free ssDNA with no RC attached.


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Figure 2. FPLC purification trace of DNA (Strand-1) conjugated RCs. Chromatographs at 260 nm (green), 280 nm (red) and 365 nm (blue) are shown. The absorbance bands at 260 nm and 280 nm are from both RC and DNA, whereas the absorbance bands at 365 nm are from the RC. The fractions from each of the peaks were collected separately and their respective absorbance spectra measured. Schematics corresponding to the absorbance spectra showing number of DNA strands conjugated per RC are given at the top of the figure. Dye-labeled pre-annealed Strand-2 and -3 are then allowed to hybridize to the purified Strand 1-conjugated-RC to create 3arm-DNA-RC conjugates with one, two or three 3arm-DNA junctions on each RC (Scheme S3-S4) carrying different identities and numbers of dyes. Cy3modified Strand-3 and Cy5-modified Strand-2 were purchased from Integrated DNA Technologies (IDTDNA). AF660-modified Strand-3 and AF750-modified Strand-2 were synthesized by reacting amine-modified DNA (Strand-2 or -3, synthesized using a DNA synthesizer) with the succinimidyl ester of the corresponding dye (purchased from Invitrogen). The resulting conjugate was subsequently purified by reverse phase HPLC and characterized using

matrix-assisted

laser

desorption

ionization

time-of-flight

(MALDI-TOF)

mass

spectroscopy (see details in the Supporting Information, Figure S1).

Table 1. 3arm-to-RC ratio of different constructs Dye Cy3/Cy5

AF660/AF750

Sample 3arm-Cy3-RC(1DNA) 3arm-Cy3-RC(2DNA) 3arm-Cy3-RC(3DNA) 3arm-Cy3-Cy5-RC(1DNA) 3arm-Cy3-Cy5-RC(2DNA) 3arm-Cy3-Cy5-RC(3DNA) 3arm-660-RC(1DNA) 3arm-660-RC(2DNA) 3arm-660-RC(3DNA) 3arm-660-750-RC(1DNA) 3arm-660-750-RC(2DNA) 3arm-660-750-RC(3DNA)

Abbreviation 1C 2C 3C 1CC 2CC 3CC 1-6 2-6 3-6 1-6-7 2-6-7 3-6-7


3arm/RCa 0.75 ±0.05 1.65 ±0.05 2.35 ±0.05 0.8 ±0 1.65 ±0.05 2.2 ±0.1 0.85 ±0.15 1.6 ±0 2.15 ±0.05 0.9 ±0.1 1.65 ±0.05 2.0 ±0.1

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a

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The molar ratios of the 3arm/RC were obtained by measuring the dye concentration and the RC

concentration, calculated from their UV-vis absorbance spectra and known absorption coefficients, assuming a 100% dye labeling ratio on the HPLC purified DNA strands.

Figure 3: Absorption spectra of representative 3arm-DNA-dye-RC constructs. (A) Absorption spectra of RC, 3C and 3CC (B) Absorption spectra of RC, 3-6 and 3-6-7.

The assembly of the 3arm-DNA-RC constructs containing only Cy3 and different DNA/RC ratios are named 1C, 2C or 3C (Abbreviations as in Table 1). These were created by assembling Strand-2 (unmodified) and Cy3-modified Strand-3 with the FPLC fractions that contained conjugates of one, two or three Strand-1 conjugates per RC. The spectra of these structures show enhanced absorbance between 450-580 nm compared to the RC alone, due to the additional absorbance from Cy3 in this spectral region (Figures 3A and S5). 3arm-DNA nanostructure-to-RC ratios of 0.75 ±0.05, 1.65 ±0.05, and 2.35 ±0.05 were calculated based on the UV-vis absorbance spectra for 1C, 2C and 3C (see note in Table 1 caption). Apparently, the yield of assembly for the fully loaded 3arm-DNA junction on the RC was ~75-80%. This

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